Escherichia coli O157:H7 (EHEC) is a food-borne pathogen that causes bloody diarrhea and hemolytic uremic syndrome (HUS), a complication of EHEC disease that can lead to kidney failure, throughout the world. EHEC has a very low infectious dose, making it difficult to control epidemiologically. EHEC colonizes the large intestine where it attaches to colonic epithelial cells and causes effacement of microvilli. EHEC also produces Shiga toxin that causes the major symptoms of HUS. In order to successfully colonize the host and establish infection, EHEC must be able to compete with the indigenous microbiota for nutrients. Ethanolamine (EA) is present in the gastrointestinal tract. EHEC uses EA as a nitrogen source, thereby gaining a competitive advantage over the indigenous micriobiota. Importantly, EHEC has co-opted EA as a signal to promote expression of genes involved in host colonization and infection as well as in Shiga toxin production. The transcriptional regulator EutR activates expression of genes that code for EA metabolism, and we determined that EutR promotes expression of EHEC virulence genes. Although EutR is important for EHEC pathogenesis, very little is known concerning the extent of EutR gene regulation in EHEC or how EutR functions to regulate virulence genes. In Specific Aim 1, we will perform a detailed characterization of the role of EutR in EHEC pathogenesis as well as examine the mechanisms of EutR virulence gene regulation. Interestingly, EHEC responds to EA and modulates virulence gene expression in the eutR mutant strain suggesting that EHEC encodes a second EA sensor. In Specific Aim 2, we will investigate the roles and mechanisms of EA-dependent, EutR-independent virulence gene regulation in EHEC. Specific Aim 3 will address how these EA-sensors contribute to EHEC fitness and virulence gene expression during infection. The proposed experimental approaches will achieve a better understanding of mechanisms used by EHEC to activate its virulence genes and may reveal novel aspects of EHEC pathogenesis. Altogether, these data may lead to the development of unique strategies for intervention and/or treatment for EHEC disease.